1. Field of the Invention
The present invention is directed to a device for determining high density cholesterol (HDL) which allows the user to obtain rapid, reliable results in a simple manner. More specifically, the present invention is directed to HDL test strips utilizing dry chemistry.
2. Prior Art
Measurement of high density cholesterol, particularly in conjunction with cholesterol measurement, has been proven to be an effective indicator of potential risk to atherosclerotic cardiovascular disease (CVD). Therefore, the determination of high density lipoprotein (HDL) cholesterol has become important and common in clinical laboratories.
The traditional method of measuring these analytes is via wet chemistry, although commercial dry chemistry tests are available for total cholesterol. HDL cholesterol measurements, however, continue to be time consuming.
For a measurement of high density cholesterol, one needs to separate serum/plasma from whole blood by traditional methods of clotting or centrifugation. The separated plasma or serum is then added in a precise ratio with a precipitant system and mixed thoroughly. The plasma/precipitant mixture is allowed to sit for 5-25 minutes to allow the completion of precipitate formation and agglomeration of the precipitated particles. After this step, the mixture is centrifuged to allow the precipitate to form a cake at the bottom of the centrifuge tube and the supernatant containing high density lipoprotein (HDL) is carefully withdrawn. The cholesterol associated with this HDL fraction (HDL cholesterol) is then measured either via wet chemistry or could be measured by a cholesterol dry strip if designed to do so.
Another common method used for HDL cholesterol measurement is ultracentrifugation wherein various cholesterol-containing fractions are separated in an ultracentrifuge. This method is even more laborious and time consuming and requires considerable technical skill. Only a few reference laboratories are equipped to measure HDL cholesterol in this way and this method is quite expensive. Electrophoresis of lipoproteins has also been used but this again is slow, expensive and semi-quantitative. It is usually used only as an adjunct to other quantitative methods.
HDL cholesterol measurements therefore tend to be time consuming with manual methods. These steps can be automated, and for a large volume of sample throughput as in many clinical laboratories, analyzers which can dispense and process the reagents automatically are available but can be quite complex and expensive.
Many patents have been issued which describe the various physical arrangements for blood testing. These include systems which involve lateral or horizontal movement of the blood, as well as plasma testing. For example, U.S. Pat. No. 4,876,067 to Deneke et al; U.S. Pat. Nos. 4,861,712 to Bartl et al; 4,839,297 to Freitag et al and U.S. Pat. No. 4,786,603 to Wielinger et al, all assigned to Boehringer Mannheim GmbH, describe test carriers and methods for analytical determination of components of bodily fluids, including separating plasma from blood using glass fibers and the like. These patents, however, all teach systems which require some type of rotation of test pads or a portion of the test pads during use. U.S. Pat. No. 4,816,224 to Vogel et al also assigned to Boehringer Mannheim GmbH, describes a device for separating plasma or serum from whole blood and analyzing the serum using a glass fiber layer having specific dimensions and absorption to separate out the plasma from the whole blood for subsequent reaction.
U.S. Pat. No. 4,857,453 to Ullman et al describes a device for performing an assay using capillary action and a test strip containing sealed liquid reagents including visible indicators. U.S. Pat. No. 4,906,439 to Grenner describes a diagnostic device for efficiently and accurately analyzing a sample of bodily fluid using fluid delivery in a lateral movement via flow through channels or grooves.
In addition to the above patents which are representative of the prior art showing various physical types of systems for blood testing and the like, recent patents have issued which are directed to the particular chemistry for the determination of HDL cholesterol. Thus, U.S. Pat. Nos. 4,851,335 to Kerscher et al and U.S. Pat. No. 4,892,815 also to Kerscher et al, describe specific types of processes and reagents for HDL cholesterol determination. These inventions take advantage of the different reactivities of HDL cholesterol versus cholesterol contained in other low and very low density lipoproteins (LDL and VLDL). These inventions thereby eliminate the traditional separation steps necessary for HDL cholesterol determination. The measurements are kinetic, meaning the rate of reaction of HDL cholesterol is monitored after LDL and VLDL cholesterol have all been reacted and requires careful control of time and temperature. Precisely controlled volumes of reagents are added at precise times in a prescribed manner. Measurement times are 3-10 minutes (U.S. Pat. No. 4,892,815). Even though this presents a significant improvement, for accurate results, it needs careful operator supervision if done manually or expensive instrumentation if automated.
Notwithstanding all of the above prior art, there has been no simple combined physical system and chemistry which enables the user to simply, efficiently and quickly obtain HDL cholesterol readings.
The present invention describes another approach, wherein the sample processing, including plasma separation, precipitant metering, precipitate separation as well as HDL cholesterol reactions are built into a strip such that user manipulations are minimized and HDL cholesterol can be measured in one to two minutes directly from whole blood. The method measures the end-point of the reaction and therefore precise time and temperature controls are not necessary. This method uses a device similar to that described in the parent application (of which this is a Continuation-in-Part, fully referenced and incorporated herein above) for separation of plasma and for measurement of cholesterol, except that specific dry chemistry for HDL determination is used. The device employs a tangential flow of blood across the blood cell separation membrane. HDL dry chemistry precipitation reagents as well as precipitate filters are built into the present invention device.